Dendritic cells (DC) are antigen-presenting cells specialized in efficiently stimulating T cells, and it is because of this that they are fundamental for inducing a specific immune response. Immature DCs are strategically distributed in tissues and organs where they act as sentinels, continuously taking antigen samples in their micro-environment. DCs start a maturation process as a response to different stimuli, including antigen themselves, microorganism products and tissue danger signals, which leads to migration towards T areas of secondary lymphatic organs, carrying the antigens loaded by them in the periphery. Here, mature DCs, showing an increase in the expression of HLA-II and co-stimulatory molecules, interact with T-cells presenting the antigenic peptides to them after these are processed, and stimulating them to initiate the specific response.
Immature DCs capture antigens by receptor-mediated endocytosis, micropinocytosis and phagocytosis. There are different receptors involved in endocytic uptake, among which mannose receptors are found (MR) (CD206 and CD209). They recognize terminal mannose residues, fucose and/or N-acetylglucosamine through carbohydrate recognising domains. Natural ligands include products of bacterial origin (glycoproteins and glycolipids), as well as mammal glycoproteins having high mannose content. MRs are expressed in macrophages and immature DCs, being involved in the endocytosis of mannosylated antigens for the processing and T-cell presentation thereof.
Immunotherapy in IgE antibodies-mediated allergic conditions is based on administration of therapeutic vaccines whose antigens are the same allergens as those the allergic patient is sensitised to. Allergens are proteins from pollens, dust mites, epithelium, etc, which are the superstructures carried in the air the patient breaths (environmental inhalants). It is widely accepted that the clinical efficacy of these vaccines is associated to the allergen dose delivered, so the WHO and consensus guidelines from scientific societies recommend that vaccines should be prepared with a sufficient allergen concentration. This requirement implies that the allergic patient response to the vaccine dose involves the risk of adverse effects which are intended to be limited. A way to avoid this is the preparation of vaccines based on modified allergens (allergoids) showing less capacity of reaction against IgE antibodies (less allergenicity).
Chemical modifications based on treating allergens with formaldehyde and/or glutaraldehyde, are the most widely used among vaccine manufacturers. Reaction of aldehyde groups (R—CHO) with amino groups (R—NH2) which are present in allergens amino acids, for example lysines, is the basis of such modification. Contrary to formaldehyde, which is a mono-aldehyde, the glutaraldehyde is a dialdehyde having two R—CHO groups capable of reacting with lysines R—NH2 present in different molecules. This causes an allergen polymerization and a loss in reactivity with specific IgE antibodies (the allergoid which is formed by means of formaldehyde is based on the structural modification of the protein, not on the polymerization thereof). This polymerization is considered to determine the allergoid lower allergenicity, since the IgE antibodies lose their accessibility to react with their epitopes (allergen binding sites), and reduce the number of mast cells sensitised with IgE which can be activated by them. Loss of allergenicity of polymerized allergens may imply a loss in immunogenicity, which would reduce the clinical efficacy of these preparations. It has been suggested that polymerization reduces the access of MR in DCs to the allergen mannose residues, and that this is decisive for polymerized allergens loss of immunogenicity. This is supported by the fact that the mannose residues are one of the main ligands used by DCs for allergens uptake, and that these cells play a critical role in allergen presentation to responding T cells.
Methods for the conjugation of sugars to proteins are mainly based in activating sugar by an oxidation process, generating reactive aldehyde groups (R—CHO) by conversion of the cis-glycol groups. The R—CHO generated in the oxidized sugar, after treatment with metaperiodate for example, may react with the ε-amino groups of lysines, resulting in the formation of Schiff bases. This methodology is highly appropriate for conjugating glycoproteins (e.g., enzymes, antibodies), since their carbohydrate residue is used for conjugation and avoids the protein portion associated to the biological activity thereof.
Although sugar oxidation with periodate has also been reported as a way to mannosylate proteins (Masarova et al. 2002. Int. J. Polymer. Anal. Charact., 7: 106-116), including allergens (Weinberger et al. 2013. J Control Release, 165: 101-109), its use to mannosylate polymerized antigens has two major drawbacks:                a) The oxidation of sugars produces the rupture of bonds between adjacent carbon atoms which contain hydroxyl groups (OH) and which are the basis for reactive aldehydes generation. Said rupture affects the mannose structure (Shibuya, N., et al. 1988. J. Biol. Chem., 263: 728-734), altering its capacity of binding to mannose-recognising lectins (Masarova et al. 2001, Chem. Pap. 55: 130-135) and its DC activation capacity (Sheng et al, 2006. Immunology, 118:372-383). Although the loss of mannose structural integrity may be minimized by reducing the degree of oxidation (Masarova et al. 2001, Chem. Pap. 55: 130-135), its efficiency for conjugation under milder conditions is subjected to the nature of the protein to be mannosylated (Weinberger et al. 2013, J. Control Release, 165: 101-109). In order to preserve mannose in the native form thereof, there have been attempts to perform mannosylation of proteins by means of glycosylation reactions at high temperature, but with a negative result in the absence of oxidation (Kanska et al. 2008. Biotechnol. Appl. Biochem. 49: 57-64).        b) Mannose activated after its oxidation generates reactive aldehydes which must interact with the free amino groups of the protein to be mannosylated. However, polymerization of proteins with glutaraldehyde produces a dramatic reduction of these amino groups, since they have already been used in their reaction with glutaraldehyde itself. Under these conditions, the efficiency of mannose activated to mannosylate proteins previously treated with glutaraldehyde is likely to be very low, as it is glutaraldehyde capacity to polymerize when lacking the amino groups to which it may bind. (Silva et al. 2004. Food Technol. Biotechnol. 42: 51-56). This inconvenient does not only affect mannosylation of polymerized allergens, but also that of any protein polymerized with gutaraldehyde which might eventually be intended to be mannosylated.        
Patterson et al. 1977 (J Allergy Clinical Immunology 59: 314-319) describe allergen polymerization (gramineae pollen) with glutaraldehyde. The polymerized allergens are hypoallergenic since they show reduced capacity of activation of mastocytes sensitised with IgE antibodies.
Subiza et al. 2008 (Clinical and Experimental Allergy, 39: 987-994) describe the use of allergens (gramineae pollen, Trisetum paniceum and Dactylis glomerata) modified with glutaralehyde (also referred to as allergoids) for immunotherapy in allergy. Researchers report that vaccination with allergoids from gramineae obtained by polymerization with glutaraldehyde is effective.
Heydenreich et al. 2012 (Immunology, 136: 208-217) describe a comparative study of differences in immunogenicity and allergenicity between allergen extracts from intact pollen from Phleum pratense and Betula verrucosa species, and their corresponding allergoids modified with glutaraldehyde or formaldehyde. It is reported that modification with glutaraldehyde reduces allergenicity and immunogenicity of allergoids more than modification with formaldehyde, and that DCs do not capture this type of modified allergens efficiently.
Weinberger et al. 2013 (Journal of Control Release, 165: 101-109) describe conjugation of allergenic proteins (ovalbumin and papain) to mannose activating the sugar by mild oxidation with periodate. A different degree of efficiency is obtained depending on the protein to be mannosylated. The mannosylated conjugates are reported to be captured by DCs in vivo and to produce an immune response in mice, so they could be useful for immunotherapy.
Therefore, in the state of the art there is a need to provide a method for obtaining vaccines based on polymerized and mannosylated antigens which is alternative to those currently used in the state of the art, and which allows antigen polymerization as well as its conjugation to mannose in a highly effective way, without the sugar losing its structural integrity and without the polymer properties (lower allergenicity) being affected, so that the vaccine, based on polymerized and mannosylated proteins, increase the immunogenicity thereof by improving their uptake by DCs.